A potential cure for tumor‐associated immunosuppression by Toxoplasma gondii

Abstract Background Recently, immunotherapy has become very hopeful for cancer therapy. Cancer treatment through immunotherapy has excellent specificity and less toxicity than conventional chemoradiotherapy. Pathogens have been used in cancer immunotherapy for a long time. The current study aims to evaluate the possibility of Toxoplasma gondii (T. gondii) as a probable treatment for cancers such as melanoma, breast, ovarian, lung, and pancreatic cancer. Recent findings Nonreplicating type I uracil auxotrophic mutants of T. gondii can stimulate immune responses against tumors by reverse immunosuppression at the cellular level. T. gondii can be utilized to research T helper 1 (Th1) cell immunity in intracellular infections. Avirulent T. gondii uracil auxotroph vaccine can change the tumor's immunosuppression and improve the production of type 1 helper cell cytokines, i.e., Interferon‐gamma (IFN‐γ) and Interleukin‐12 (IL‐12) and activate tumor‐related Cluster of Differentiation 8 (CD8+) T cells to identify and destroy cancer cells. The T. gondii profilin protein, along with T. gondii secreted proteins, have been found to exhibit promising properties in the treatment of various cancers. These proteins are being studied for their potential to inhibit tumor growth and enhance the effectiveness of cancer therapies. Their unique mechanisms of action make them valuable candidates for targeted interventions in ovarian cancer, breast cancer, pancreatic cancer, melanoma, and lung cancer treatments. Conclusion In summary, the study underscores the significant potential of harnessing T. gondii, including its diverse array of proteins and antigens, particularly in its avirulent form, as a groundbreaking approach in cancer immunotherapy.

advanced-stage cancers, surgery may not be a viable option, even if it is effective for localized tumors. 5The advent of immunotherapy, on the other hand, has instilled fresh optimism in cancer treatment. 6Through immunotherapy, the immune system is stimulated to fight off cancer cells. 7In comparison with conventional treatments, this approach is more specific in targeting tumor cells and offers less toxicity. 8By utilizing the potential of the immune system, immunotherapy seeks to enhance the body's inherent ability to combat cancer cells. 9,10Using these therapies, the immune system is stimulated or manipulated so that cancer cells can be recognized and destroyed more effectively by the immune system.In addition to having shown remarkable success in treating some types of cancer, immunotherapy has proven to be variable in its efficiency among different patients, and research continues to be conducted to improve its results. 11,12ploying pathogenic microorganisms in cancer immunotherapy is not new.Serratia spp, 13 Streptococcus spp, 14 Listeria monocytogenes, 15 Escherichia coli, 16 Lactobacillus casei, 17 Oncolytic virus, 18 Talimogene laherparepve, 19 Newcastle disease virus, 20 yellow fever vaccine 17D, 21 Trypanosoma cruzi, 22 Echinococcus granulosus, 23,24 and Trichinella spiralis, 25 have been shown to eradicate tumor cells. 26Toxoplasma gondii (T.gondii) can regulate innate immune cells by invading myeloid cells, typically producing powerful Th1 immune responses. 27T. gondii secretes a collection of effector molecules during the invasion, allowing it to control the host cells. 28,29The parasite even attacks adjacent cells that are reached but not invaded.For example, T. gondii activates the ROP16-mediated STAT3 and STAT6 signaling pathways in macrophages, leading to downregulation of IL-12 production and induction of arginase 1, respectively. 30,31T. gondii release polymorphic rhoptry protein kinase ROP18 as well. 32This protein protects the parasitecontaining vacuoles against the host's innate immunity mediated by IFN-activated GTPases. 29,33,34T. gondii, as a eukaryotic microbe, does not produce significant toxins or other toxic molecules, unlike many prokaryotic microbes. 35A recent study developed a stable and safe single-copy variant of T. gondii as an uracil auxotroph (cps) strain. 36During the invasion of mammalian cells without uracil, the virulence of the cps strain was significantly reduced in both normal and severely immunodeficient mice. 37This engineered uracil auxotroph elicited strongly polarized Th1 host responses that could enhance the activity of innate immune cells in the tumor microenvironment and lead to tumor regression.
In an analysis of 150 cancer patients and 120 normal people without cancer, it was observed that individuals with low levels of anti-T.gondii antibodies may have a more favorable prognosis. 38This suggests that an asymptomatic T. gondii infection has the potential to stimulate the immune system's response against tumors.Transcriptomics data indicated that T. gondii influences gene regulation, particularly in pathways associated with breast cancer, colorectal cancer, and non-small cell lung cancer. 39This interference may ultimately impede tumor growth.Further, T. gondii infection appears to inhibit tumor growth and its invasion in many cancers.Hypoxia, avascular necrosis, suppression of tumor angiogenesis, and induction of Th1 immunity have all been proposed as potential mechanisms why T. gondii has antitumor effects. 40,41The underlying reason for these effects appears to stem from the similarity between the molecular signaling pathways regulated by the parasite and those disrupted during the development of cancer.As an example, T. gondii infection can transform natural killer (NK) cells into ILC1 cells within the tumor microenvironment (TME). 42This phenomenon signifies the presence of a cell population with immune memory capabilities that can circulate throughout the body and continue producing IFN-g.
Recombinant immunogenic proteins and T. gondii extracts are used to generate vaccines containing T. gondii antigens from an array of species (Figure 1).To investigate the potential immunotherapeutic benefits of T. gondii in cancer treatment, the current study has compiled a summary detailing the mechanisms through which T. gondii infection demonstrates an anti-tumor impact.Furthermore, the present study focused on attenuated strains of T. gondii and its proteins and the activation of anti-tumor signaling within the host triggered by T. gondii infection.The current review aimed to evaluate T. gondii as a possible treatment for melanoma, breast, ovarian, lung, prostate, and pancreatic cancers.Furthermore, the potential of combining cancer immunotherapy and nanotechnology is explained.

| ROLE OF CYTOKINES AND IMMUNE RESPONSES IN ANTICANCER MECHANISMS
When infected with pathogens, dendritic cells (DCs) improve their ability to present antigens, secrete anti-tumor cytokines, and activate innate immune responses in TMEs. 43Various immune cells, including antigen-presenting cells (APCs), NK cells, CD4 and CD8+ lymphocytes, and B-cells, have been found to produce and release IFN-γ. 44,45is cytokine has the ability to trigger macrophages and the expression of major histocompatibility complex (MHC) class I or II, as well as APC's costimulatory molecules. 46N-γ and IFNα have several similar biological functions, including inducing signaling pathways that inhibit cell proliferation and promote the expression of MHC class I molecules.47,48 Additionally, IFNα impedes the advancement of cancer.It induces apoptosis in a caspasedependent manner, resulting in anti-angiogenic effects and enhancing the cytotoxicity and survival of NK cells, ultimately leading to tumor cell apoptosis.49 Another significant cytokine, IL-12, is predominantly secreted by APCs in response to pathogens.It plays a crucial role in promoting cell-mediated immunity and has been shown to drive the polarization of Th1 lymphocytes, increase NK and T lymphocyte activity, and elevate the expression of IFN-γ.50 In in vivo, infection of T. gondii with macrophages can suppress the growth of tumor cells in mice with immune-deficient systems, and in-vitro infection can activate macrophages to kill cancer cells.[51][52][53] The effectiveness of Non-Replicating Toxoplasma Uracil Auxotrophs (NRTUAs) in combating tumors is diminished when CD8+ and NK cells are depleted, while the presence of CD4+ cells does not have the same impact. 54 Stdies have indicated that CD8+ T cells and CD8+ T cells are essential for achieving best anti-cancer responses.Following NRTUA vaccination, there is a rapid increase in CD19+ B cells as well as CD3+ and CD8+ T cells at the site of inoculation.55 Subsequently, a vigorous Th1 immune response was initiated, demonstrating the substantial potential of these cells in triggering a mutually beneficial immune response against tumors.All of these findings provide evidence that the suppression of tumors by NRTUAs relies on intact immunity, particularly the CD8-dependent immunity.In addition to triggering CD8+ T cell-dependent immunity, NRTUA infection also stimulates Th2 humoral immunity against cancer cells.56 In a murine model of pancreatic cancer (Pan02), NRTUA treatment stimulates the production of tumor-specific IgG.Additionally, CD4+ T cells have been found to have an important function in the response to a subsequent challenge with Pan02. 57Overall, an enduring CD8 + -dependent immune response may be established with NRTUA therapy.An infection with T. gondii activates innate immunity to produce IL-12, which promotes the T cells and NK cells' anti-tumor responses by facilitating the polarization of M1 macrophages, inducing the expression of IFN-g, and inhibiting angiogenesis.58,59 Mice that lack functional DCs exhibit a notable shortage of IL-12 and are more susceptible to T. gondii.60 Moreover, the IL-12 triggered by T. gondii relies on the activation of CD8+ T cells through Toll-like receptor (TLR) signaling.61 A significant elevation in the serum levels of IL-12p40 and IL-12p70 has been observed in mice with ovarian cancer, melanoma, and pancreatic cancer after administration of NRTUAs as vaccination.54 The treatment with RH-Dcps, which activates myeloid cells, lost its ability to fight tumors in mice that lacked IL-12p40 or IL-12p35.26 This suggests that the production of IL-12 by NRTUA-stimulated cells is crucial for anti-cancer immunity.Of note, there are two distinct immunological phases observed in the treatment of melanoma.In the initial stage, the presence of host IL-12 is not necessary, but in the subsequent phase, it needs to be expressed by DCs and macrophages.62 While secretion of IL-12 which is influenced by the presence of MYD88, is indispensable in resisting T. gondii infection, it is not essential for eliciting an anti-cancer response in instances of melanoma and ovarian cancer.63 An effective anti-tumor response is dependent on IL-12 in pancreatic cancer. A ack of IL-12 in the body may make it difficult to eliminate malignant tumors and T. gondii infection (Figure 2).

| TOXOPLASMA GONDII PROFILIN PROTEIN
Actin-binding T. gondii profilin (Tgprofilin) with the molecular weight of 14-19 kDa was found in Soluble T. gondii antigens (STAg). 64It enhances the immune response and lessens the pathogenic effects of bacteria, viruses, and other diseases-causing agents. 65The profilin-like protein (TgPLP) has a molecular weight of 17.5 kD and a high degree of similarity to Tgprofilin. 66,67The distinction between Tgprofilin and TgPLP is not always clear in some researches.As a result, TgPLP is currently considered a component of Tgprofilin.Tgprofilin serves F I G U R E 1 An overview of various approaches to developing toxoplasmosis vaccines is presented in this figure.The infection process of Toxoplasma gondii consists of three distinct stages: tachyzoites, bradyzoites (cysts), and sporozoites (oocysts).Regardless of the stage of T. gondii, multiple bradyzoite/sporozoite antigens can be expressed on tachyzoites to enhance live vaccines' immunogenicity.Recombinant immunogenic proteins and T. gondii extracts are used to generate vaccines containing T. gondii antigens from an array of species.Delivery systems and adjuvants are crucial for improving subunit vaccine effectiveness by enhancing antigen availability and enhancing immune responses.It is necessary to conduct further research to identify specific T. gondii epitopes that target CD4+, CD8+, and B cells.An epitope vaccine that elicits robust T and B cell responses needs to be designed rationally based on this knowledge.A vaccine based on these epitopes would broaden the spectrum of immune responses induced by antigens to a wide range.multiple functions, including contributing to T. gondii's movement and attacking host cells.Moreover, through the activation of MyD88, it also stimulates the production of IL-12 by acting as an agonist for TLR11. 66 a supplementary mechanism, TgPLP stimulates MyD88 signaling pathway during autologous whole-tumor-cell vaccine therapy.Macrophages derived from bone marrow express more antigen-presenting cell biomarkers as a result of this activation.As a result, the production of IL-12 is enhanced, improving the macrophages' ability to engulf cancer cells. 68spite profilin or STAg administration, IFN-g deficient mice (IFN-g À/À) were unable to effectively suppress pancreatic tumor growth.This suggests that pancreatic cancer treatment requires DCs and IFN-g.

| TOXOPLASMA GONDII SECRETED PROTEINS
A tachyzoite culture medium contains excretory/secretory proteins (ESP) released by T. gondii. 69In individuals who have been previously infected, these proteins contain elements that attach specifically to serum antibodies, as well as components that act as proteases. 70As with TLA components, ESPs pose the primary candidates for potential vaccines with anti-tumor properties. 71Various cell lines, including MCF-7 breast cancer cells, K562 erythroleukemic cells, and DU145 prostate cancer cells, exhibit apoptosis induced by T. gondii ESP. 72ese cells were exposed to varying concentrations of T. gondii tachyzoites during the mid-exponential phase.The findings revealed a remarkable dose-dependent inhibitory impact of the parasite on cancer cell development.Moreover, the expression of p53 protein in A549 tumor cells was increased while the expression of anti-apoptotic Bcl-2 gene was down-regulated following treatment with T. gondii.Additionally, T. gondii ESP prevents Lewis lung carcinoma, B16F10 melanoma, and prostate cancer cells from growing.It has been shown that the administration of ESP to mice after subcutaneous injection of Lewis lung cancer cells and B16F10 melanoma cells in the right armpit resulted in a significant decrease in Treg of CD4 + CD25 + Foxp3+ and an increase in NK cells in the spleen. 73Furthermore, ESP-treated subjects had significantly smaller tumors than control subjects. 74HPLC analysis confirms the presence of dense granule antigen 2 (GRA2) and GRA5, among other proteins, in ESP components. 75For effective invasion, T. gondii produces proteins sequentially from its secretory organelles, which include Microneme Proteins (MICs), Rhoptry Proteins (ROPs), and Gross Adhesion Proteins (GRAs). 76,77Signaling pathways are also regulated by these proteins.To facilitate parasite proliferation and viability, T. gondii releases specific proteins that are attracted to the parasitophorous vacuole membrane (PVM). 78These proteins are then recognized by host cells, triggering an immune response against the tumor.The removal of PVM-related proteins as well as intra-vacuolar network-related GRAs diminishes anti-cancer immunity. 79,80Specifically, when rop5 (Drop5) was abrogated, the anti-cancer immunity in IFN-g-induced mouse fibroblasts was severely compromised. 81By introducing a new synthesized GRA8 peptide, the exporting of GRA8 to mitochondria, its interplay with deacetylase (sirtuin-3), and controlling of mitochondrial function are enhanced. 82This enhancement ultimately results in higher effectiveness of mitochondria-targeted therapies for HCT116 tumors.However, the removal of GRA3 and ROP12 does not hinder the anti-cancer impact of the microorganism.
Treatment of lung carcinoma, melanoma, and colon adenocarcinoma with a combination of T. gondii type I ΔGRA17 strain with the concentration of 10 6 tachyzoites and 250 μg PD-L1 inhibitors demonstrated notable efficacy. 83This approach enhances the innate and adaptive CD4+ T cells, preventing immunosuppressive pathways in TME.They also demonstrated that intertumoral administration of ΔGRA17 led to the infiltration of immune cells in both injected and distant tumors.This significantly improved the survival of mouse models without any evidence of the recurrence of their disease.DNA damage and signals from oncogenes can stimulate the expression of gene p53.PVM transports dense granule-secreted GRA16 to the nucleus, where it alters host gene transcription. 84GRA16 interacts with the host proteins such as protein phosphatase 2A and ubiquitin-specific protease 7.This interaction allows GRA16 to affect the progression of the cell cycle and restore p53 function.Five specific sequences within the host cell's nucleus were involved in this restoration.Additionally, GRA16 enhances the effectiveness of irinotecan, a chemotherapy drug, by suppressing the activity of NF-kB in lung H1299 cancer cells. 85It is known that NF-kB plays a role in immunosurveillance of cancer cells.In addition, it controls transcription factor communication (e.g., TRAFs), regulating cell proliferation, apoptosis, and the release of inflammatory cytokines. 86,87(Figure 3).

| OVARIAN CANCER
Ovarian cancer is the deadliest female reproductive cancer. 88Due to its generally vague symptoms, this cancer is often not detected until it has reached an advanced stage, making effective treatment difficult. 89s are the most prevalent leukocyte subpopulation in ovarian carcinoma and develop a highly immunosuppressive tumor environment at tumor sites.The infiltration of immature CD11c + dendritic cells in solid epithelial ovarian tumors positively contributed to releasing various proangiogenic and immunosuppressive mediators. 90,91A previous study, found that ovarian cancer-infiltrating DCs display a classical phenotype but express high levels of PD-1 and B7-H1 on their surface. 92These cells inhibit the accumulation and functioning of CD8 + T cells within the tumor microenvironment and block Nuclear Factor Kappa B (NF-kB) activation.Therefore, any successful immunotherapy approach for ovarian cancer must overcome the tumor's immunosuppressive microenvironment. 935][96] Enhancing the antigen presentation capacity of ovarian cancer-infiltrating DCs and subsequent T-cell-driven antitumor immunity can be achieved by reversing CD11c + MHC-II + from an immunosuppressive to an immunostimulatory phenotype.
Intertumoral treatment with nonreplicating cps strain of T. gondii is demonstrated to reverse the tumor-associated immunosuppression of CD11c + APCs into immunostimulatory status. 37While resistance to T. gondii depends on MyD88-mediated IL-12 production, it is not crucial for the anti-tumor responses of ovarian cancer.Fox and colleagues showed that in addition to activating host cell invasion, uracil auxotrophic mutants of T. gondii provide efficient antitumor immunity against ID8DV ovarian cancer through the secretion of vacuole

| BREAST CANCER
Women were diagnosed with breast cancer more frequently than men in 2020, with an estimated 2.3 million new patients. 97Cancerous cells that lack human epidermal growth factor receptor-2 (HER2), estrogen receptor (ER), and progesterone receptor (PR) are referred to as triplenegative breast carcinomas (TNBC). 98TNBC is the most prevalent malignant breast cancer subtype.It has the worst prognosis among all the carcinomas. 99These days, medical procedures, radiation treatment, and chemotherapy are vital choices for treating malignant breast cancer routinely, and chemotherapy continues to be the backbone of TNBC therapy. 100Even though many new treatments have been assessed, the impacts of treating TNBC remain poor, emphasizing the need for a novel technique for viable TNBC treatment. 101Cancer vaccines have attracted a legion of attention as a way forward for cancer treatment.
Nevertheless, the unfortunate immunogenicity of cancer-related antigens often fails to elicit a productive and safe response against the tumor. 102[105] Thus, parasite antigens with high immunogenicity and significant epitope similarity with breast cancer antigens may stimulate a robust and safe response against many tumor cells. 105,106T. gondii can trigger an immune response caused by the release of IFN-γ and IL-12, similar to those against breast cancer. 107Inoculating an attenuated uracil auxotroph T. gondii vaccine might inhibit breast cancer progression if the parasite infection affects tumor-related factors. 51Scientists hypothesize that infection with T. gondii may influence the breast cancer signaling pathway. 108The anti-tumor potential of the avirulent uracil auxotroph RH-Δompdc mutant was experimentally evaluated by Xu et al. 108 They reasoned that intratumoral administration of these engineered live attenuated parasites into 4T1 breast tumor caused tumor regression and decreased lung metastasis.Moreover, the tumorbearing mice experienced a more prolonged survival rate.It is believed that the IL-12 signaling pathway regulates these results.Monocytes, the earliest immune cells that secrete IL-12 during T. gondii infection, trigger the production of more IL-12 from DCs and neutrophils.In turn, the IL-12 enhances the influx of NK and CD8 + T cells and secretion of IFN-γ in both serum and tumor microenvironment. 109The secreted IFN-γ eventually promotes cell-mediated immunity against tumors by inducing apoptosis in cancer cells and preventing angiogenesis in tumor tissue. 110

| PANCREATIC CANCER
Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers, with an average survival time of only 4 to 6 months after diagnosis. 111Despite advances in tumor microenvironment and improvements in the production of therapies and drugs, this cancer continues to pose a significant challenge. 112Thirty percent of patients have advanced local disease, and 50% show evidence of metastatic disease at the time of diagnosis. 113Despite some progress in improving survival rates, it is still necessary to develop additional therapies and drugs that can develop immunity to more effectively treat advanced pancreatic cancer in conjunction with existing therapies. 114w therapies that provide long-term prevention against tumor recurrence and deadly metastatic pancreatic cancer will be essential to enhance patients' long-term survival.A favorable prognosis is associated with circulating CD8 + T cells in pancreatic cancer.
Myeloid cells undergo fast reprogramming to suppress or be suppressed within the pancreatic tumor microenvironment in response to cancer. 115Attenuated T. gondii vaccine strain cps blocks immune suppression in pancreatic tumors by activating tumor-dependent myeloid cells, which then trigger a tumor cell antigen-specific CD8+ T-cell immune response. 54In a similar fashion to ovarian cancer, the expression of costimulatory molecules CD80 and CD86 as well as IL12 is upregulated by tumor-associated DCs and macrophages.Although systemic administration of IL12 causes substantial toxicity in cancer patients, IL-12 secreted following cps therapy exhibits no side effects. 116Subsequently, CD8+ T cells perform their anti-tumor benefits by recognizing the pancreatic tumor-cell antigen and producing IFNγ within the TME.Unlike ovarian and melanoma cancers, cps treatment activates the MyD88 signaling pathway in pancreatic cancer cells.

According to a study by Sanders et al., 2016 et al., mice bearing
Pan02 pancreatic cancer that received cps therapy significantly increased activated CD8+ T cell infiltration into the tumor microenvironment. 57In more details, the high amount of pancreatic tumor antigens released by CD8 + effector T cells during primary cps therapy can elicit the secretion of tumor-specific IgG, along with long lasting CD4 + T cell memory.Of note, while the survival against the primary tumor does not depend on CD4 + T cells, the distribution and stability of circulating tumor-specific IgG provide a protective effect against tumor re-challenge.However, further research is needed to fully understand the relationship between memory immune cells and circulating antibodies in the mechanism of action of the cps mutant of T. gondii for cancer treatment. 54,57,117espite recent progress in treatment strategies, melanoma remains the most commonly lethal form of skin cancer. 118Cancer immunotherapies have therefore been studied as possible treatments for metastatic melanoma. 119,120A study have shown that CD8 + T cells are effective in treating melanoma.Baird et al. investigated the anti-B16F10 melanoma effects of the attenuated strain of T. gondii (cps) by intertumoral injection. 62Their study represented the first successful use of immune-based monotherapy against primary B16F10 tumors in mice, which led to tumor shrinkage.They found that cps-based monotherapy elicited a potent in vivo anti-tumor immunological response driven by IFNγ-expressing CD8 + T cells.As the T cells were activated, the dermal melanoma tumor regressed and rarely reappeared.Besides local effectiveness, this treatment induced systemic and memory anticancer immune activity.The mice that survived showed vitiligo, a sign of good outcomes that suggest cps treatment generated melanocyte-specific CD8 + T cell immune reactions. 62The systemic response made immunotherapy preferable to surgical excision that can eradicate occult metastatic lesions.To enhance efficacy, cps monotherapy can be combined with additional immunological strategies, such as adoptive T-cell treatment.The maturation of T-cells was mediated by IL-12, and the CXCR3-stimulating cytokines.Despite pancreatic tumor, the release of IFNγ by NK-cells was crucial for the successful treatment of the B16 melanoma tumor model. 57Surprisingly, it is suggested that under the presence of cps, Th1 CD4 + T cells are no longer playing their normal role through classic Th1-type cells.Instead, other cell types such as NK cells are stepping in.Indeed, like that of ID8 ovarian cancer, the therapeutic efficacy of cps therapy was not dependent on the MYD88 signaling pathway. 26 general, evidence demonstrates that cps therapy causes immediate and long-lasting leukocyte infiltration of the tumor.There have been two distinct immunological phases throughout melanoma treatment.Myeloid, rather than lymphoid, cell recruitment occurs as the initial response to the treatment.In this case, phagocytes constitute 70%-80% of T. gondii-invaded cells, while only 20%-30% of T cells and neutrophils are affected by the parasite during the beginning phase of the treatment. 121Finally, an increase in IFNγ expression by both CD8+ T cells and NK cells, as well as an increase in the number of CD8+ T cells, occurred during subsequent treatment stages, resulting in durable protection against tumor rechallenge.This represents a significant advancement in the field of melanoma immunotherapy.

| LUNG CANCER
Lung cancer is responsible for one of the highest mortality rates from cancer-related conditions worldwide. 122,123This condition is described as the uncontrolled growth of lung cells, often associated with malignant tumor growth.Lung cancers are classified into nonsmall cell lung cancers (NSCLC) and small cell lung cancers (SCLC). 124e most common form of lung cancer is non-small cell lung cancer (NSCLC). 125The advancement of medical science has simplified lung cancer treatment, but delayed diagnosis, low response rates to treatment, and limited therapeutic options remain significant obstacles. 126Several conventional treatments are available for lung cancer, including radiation therapy, chemotherapy, surgery, targeted therapies, and immunotherapy. 127In lung cancer cases that are discovered and treated at an early stage, treatments can be quite effective; their effectiveness, however, may alter according to the type of the cancer and its stage. 128Managing this condition has become more challenging as drug resistance has evolved.
Using T. gondii as a treatment strategy for lung cancer could be a promising research direction.T. gondii was investigated in a murine model to determine whether it could counter lung cancer.Based on a study findings, T. gondii infection suppresses Lewis lung carcinoma (LLC) growth in mice, exhibiting considerable tumor-suppressing activity. 129 another study, survival rates of mice given T. gondii Me49 strain orally (TG-injected) exceeded those of mice that received LLC injections. 130Furthermore, mice injected with T. gondii and LLC cells (TG/LLC-injected group) displayed even better survival rates.Some things were associated with this result, such as higher CD8+ T-cell levels, enhancement of IFN-γ mRNA expression rates, increased IgG2a levels in the serum, and increased cytotoxic T lymphocyte (CTL) activity.When these immune responses are triggered, there is a robust immune reaction against cancer.In addition, the study found that mice injected with TG/LLC and TG showed antiangiogenic properties, preventing angiogenesis (formation of new blood vessels), an essential process for tumor proliferation.Tumor growth inhibition occurs as a result of this compound's antiangiogenic properties.A further enhancement of antitumor effects in mice injected with TG/LLC was obtained when Quil-A was added as an adjuvant.However, it's important to note that this treatment approach resulted in lower levels of CD4+ and CD8+ T-cells, IgG1 and IgG2a serum titers, and IFN-γ transcripts in mice that were injected with TG/LLC rather than TG alone.T. gondii infection appears to affect immunological responses and tumor microenvironment.
Another study examined the effects of an immunostimulant composed of formalin-fixed T. gondii organisms (f-Tp). 131F-Tp was used in this experiment to inhibit the growth of tumors in C57BL/6 mice infected with T. gondii and presenting with LLC.The substances showed significant antitumor effects when f-Tp was administered intradermally to mice with tumor cells.The tumor was reduced in size, and a longer life span was observed in the mice.f-Tp induced a delayed-type hypersensitivity (DTH) response directly correlated with its antitumor activity.Interestingly, f-Tp was an antitumor even when injected directly into tumor sites at hours 1, 3, and 5 after tumor inoculation.Furthermore, live BCG injections in BCG-sensitized mice only had a significant antitumor effect when injected with tumor cells.As a result of these findings, f-Tp injection in mice with T. gondii infection could induce potent antitumor responses.
Using a mouse xenograft model, researchers discovered that GRA16-transfected H1299 cells minimized tumor size. 85

| FUTURE AND CHALLENGES
It is essential to understand that tumor-associated immunosuppression hinders cancer treatment effectiveness. 132The full potential of T. gondii as a therapeutic agent for reinvigorating the immune response against tumors must be achieved by overcoming a number of challenges.For this novel approach to be translated into a safe and practical cure for cancer-associated immunosuppression, researchers and clinicians must address several challenges and directions in the future.

| Safety and toxicity
A therapeutic agent such as T. gondii should not lead to human health problems.Studies have shown that T. gondii can boost immune responses against tumors, though it is necessary to watch out for adverse effects.Uncontrolled parasite reproduction, inflammatory processes, and unintentional tissue damage might all contribute to the problem. 133Preclinical and clinical trials must be conducted with robust safety assessments to identify and mitigate these risks.In the future, the therapeutic window needs to meet its full potential and ensure the safety and efficacy of therapeutic interventions.

| Immunological balance
The delicate balance between reactivating the immune system and avoiding autoimmune reactions must be achieved to reactivate the immune response. 134

| Resistance and adaptation
The various treatment options for cancer are noted to lose effectiveness over time, 135 and T. gondii-based therapy is no exception.One of the crucial aspects of comprehending this therapy is anticipating how tumor cells can adapt and resist it.Combination therapies, treatment cycling, and other innovative methods are available for overcoming this resistance.Cancer research is constantly evolving, and keeping up with tumor resistance mechanisms can be difficult. 136

| Personalized medicine
Individuals react differently to T. gondii-based therapy influenced by genetic, immunological, and environmental factors. 137 In recent years, nanotechnology, or the science and engineering of materials at the nanoscale, has become a rapidly advancing field that has the potential to revolutionize all sectors of human society, including healthcare. 138In diagnostics, treatment, drug delivery, and disease management, nanomaterials with dimensions between 1 and 100 nanometers offer distinct advantages over traditional medicine. 139The role of nanotechnology in targeted therapies has been one of nanotechnology's most significant contributions to medicine. 140,141Traditional medical treatments often have limited selectivity and affect healthy tissues, leading to side effects and reduced efficacy. 142There is a wide range of nanoparticles available today for delivering drugs, genes, and imaging agents to specific cells or tissues on the basis of their size and structural properties, such as liposomes, dendrimers, and nanomicelles. 143,144The ability to cross the blood-brain barrier makes nanoparticles particularly valuable in treating conditions that were previously difficult to treat, such as brain tumors. 145In addition, controlled-release nanosystems can be used to extend the exposure of drugs, resulting in increased compliance among patients and reduced frequency of administration. 1468][149] A growing number of researchers are focusing on using nanoscale technology and nanocoatings to improve the ability of T. gondii antigens and proteins. 150,151Nanotechnology can enhance antigen presentation by improving T. gondii antigen or protein uptake by antigen-presenting cells, such as DCs. 152A variety of applications and advantages can be found in nanoscale technologies. 153,154Nanoparticles possess unique properties that enable them to serve as carriers for T. gondii antigens. 155In some cases, researchers can enhance the bioavailability and longevity of these antigens by encapsulating them or coupling them with nanoparticles. 156rough this strategy, the immune system will be targeted more precisely, tumor-specific antigens will be presented more effectively, and tumors will be attacked more effectively with an immune response. 157 vaccine production, adjuvants have a significant role, and nanoformulated adjuvants offer unique advantages. 158,159Using nano-sized adjuvants may enhance immune responses via stimulation of antigenpresenting cells and stimulation of cytokine production together with antigens from T. gondii.A synergistic effect enhances the effectiveness of cancer vaccines based on T. gondii.Proteins delivery to target cells is facilitated by nanocarriers, such as liposomes, micelles, and dendrimers. 160By delivering these proteins precisely, they enhance their interaction with the host's immune system and are better able to function as cancer immunotherapies. 161Nanomaterials such as lipid bilayers or polymer shells can be used as coatings on T. gondii vaccines.These coatings serve various functions, including prolonging circulation times, preventing antigen degradation, and facilitating controlled release. 162The possibility of reducing off-target effects and overall safety profiles can be enhanced, making the approach more effective.
immune pathways.Zhu et al. discovered that intertumoral injection of ΔGRA17 enhanced CD8+ cytotoxic T-cell infiltration, which ultimately resulted in tumor regression via upregulating IFN-γ and TNF-α.Indeed, it may suppress the expression of PD-1 in CD8+ T cells and Foxp3 in F I G U R E 2 This figure provides an overview of different categories of Toxoplasma gondii vaccines used in research and development.The represented vaccine types include inactivated vaccines, excretory-secretory antigen vaccines, live attenuated vaccines, subunit vaccines, DNA vaccines, epitope vaccines, and mRNA vaccines.Each category represents a distinct approach to T. gondii vaccine development, with unique characteristics and potential applications.

F
I G U R E 3 This figure illustrates the key processes involved in reversing immunosuppression for tumor therapy through Toxoplasma gondii vaccination.Myeloid cells in the tumor stroma are initially immunosuppressive, leading to reduced dendritic cell antigen-presenting capacity and diminished immune responses.T. gondii infection, either systemically or within tumor tissues, alleviates myeloid cell immunosuppression.This triggers the activation of DCs, which modifies the tumor microenvironment by secreting IL-12, promoting Th1 immune responses.T. gondii antigens, potent IL-12 inducers via the MyD88 signaling pathway, play a crucial role.IL-12 influences natural killer (NK) cells, CD4+, and CD8+ T lymphocytes, acting as a Th1 response stimulator and an angiogenesis inhibitor.Consequently, CD8+ T cells, NK cells, and macrophages are recruited to the tumor tissue, collaborating with their secreted IL-12 and IFN-γ to eliminate tumor cells effectively.membrane (PVM) associated rhoptry effector proteins such as ROP5, ROP17, ROP18, ROP35 or ROP38, and intravacuolar network-related dense granule proteins GRA2, GRA12, and GRA24. 78A previous study by Baird et al. 2013 conducted a study on established aggressive ID8-Vegf ovarian tumors. 26They found that cps immunotherapy preferentially restored the antigen cross-presentation ability of CD11c + cells, primed new CD8 + T cell responses, and enhanced the function of suppressed CD8+ T cell responses.Tumor regression caused by cps therapy is associated with high-level expression of T-cell receptor costimulatory molecules CD80 and CD86 and IL-12 and IFN-γ, which are crucial for activating antitumor T-cell immunity.Notably, vaccination with cps strain represents a lifelong CD8 + immunity, and its effectiveness is not affected by prior exposure to T. gondii, which is prevalent in humans.Treatment with cps is also accompanied by accumulating some splenocytes, including macrophages, B cells, NK cells, and CD4+ T cells, and decreasing the number of Foxp3+ CD4+ T regulatory cells in the tumor microenvironment.
Protein phosphatase 2A-B55 activates upon induction of the B55 regulatory subunit by GRA16, causing the GWL protein level to decrease, the ENSA phosphorylation to increase, and PP2A-B55 function to be activated.Therefore, cell survival and cell cycle arrest were suppressed, as well as phosphorylation of AKT/ERK.GRA16 enhanced Irinotecan's anticancer properties, as it inhibited NF-B activity and resulted in cell cycle arrest, thereby enhancing chemotherapeutic efficacy against NSCLC.The purpose of this study was to investigate the effects of T. gondii excretory-secretory antigens (ESA) on CD4 + CD25+ Foxp3+ Treg cells in mice with LLC and to determine whether T. gondii ESA inhibits tumor growth.C57BL/6 mice were divided into PBS and Lewis groups, with LLCs injected into the latter group.A subgroup of these groups was then subdivided into those treated with ESA and those not treated with ESA.PBS and Lewis groups showed increased spleen coefficient and reductions in splenic Treg cells after ESA treatment.Additionally, ESA treatment for T. gondii retarded tumor growth, as evidenced by significantly smaller tumors in Lewis patients who received ESA treatment versus patients who did not.In mice with LLC, the ESA of T. gondii inhibited tumor growth and reduced the proportion of Treg cells.
Therapy based on T. gondii must target tumorspecific antigens while sparing healthy tissues.Innovations in immunomodulatory techniques and precise targeting mechanisms are therefore necessary.A balance must be maintained between inducing dangerous autoimmune reactions and providing effective treatments to achieve successful outcomes.T. gondii interacts with the immune system in many ways that must be fully understood.This interaction needs further research to understand how the parasite triggers immune responses and modulates tumor-specific immunity.Comprehending these mechanisms can enhance therapeutic strategies and interventions at a molecular level.
To enhance therapeutic results, it is imperative to design individualized treatment plans tailored to the unique characteristics of each patient.Treatment regimens consist of determining biomarkers and other predictive indicators for guiding treatment choices.10.5 | Long-term monitoring and follow-up To better understand how T. gondii-based treatment impacts patients' general health and immune systems over time, prolonged monitoring and follow-up studies are needed.Research on the effectiveness and durability of immune responses caused by the therapy and clinical trials for late-onset side effects must be conducted.This type of research is crucial for evaluating therapy's safety and efficacy over the long term while offering comprehensive support once treatment is completed.10.6 | Combination therapies A synergistic combination with other immunotherapy approaches or conventional cancer treatments is being explored to optimize the efficacy of T. gondii-based therapy.It is challenging to optimize treatment sequences and combinations.Identifying therapies that complement each other and offer the most excellent chance of improving treatment outcomes needs to be studied.Adding other therapies to chemotherapy may enhance its effectiveness in combatting different cancer types and resistance mechanisms.ANTIGEN DELIVERY The requirement for safety and biological compatibility is a crucial point to consider in nanotechnology applications.Their biocompatibility and toxicity must be thoroughly evaluated as a first step in establishing T. gondii-based nano therapies as clinically viable.Nanotechnology combined with T. gondii-based cancer immunotherapy is expected to be very beneficial in developing customized therapies.Clinical trials using F I G U R E 4 Dendrimers, micelles, and liposomes are excellent nano-scale carriers for antigens of Toxoplasma gondii, as they can enhance the stimulation of the immune system.
customized nanocarriers and formulations allow clinicians to optimize treatment outcomes, ensuring that each patient receives an effective and highly personalized treatment plan (Figure4).
12 | CONCLUSIONCancer immunotherapy has made remarkable advances in activating immune responses to eradicate tumors.An impressive list of cancers that T. gondii has been documented to alleviate includes melanoma, pancreatic, breast, lung, and ovarian cancers.Treatments that stimulate similar cytokines, chemokines, and immune responses to those used to treat cancer have a high effectiveness rate.Cancer immunotherapy has made remarkable advances in activating immune responses to eradicate tumors.An impressive list of cancers that T. gondii has been documented to alleviate include melanoma, pancreatic, breast, lung, and ovarian cancers.Treatments that stimulate similar cytokines, chemokines, and immune responses to those used to treat cancer have a high effectiveness rate.In the case of tumor-associated immunosuppression, T. gondii may prove beneficial, but complex challenges must be overcome.Even though this innovative approach can reinvigorate the immune response against cancer, safety concerns, immunological balance, resistance mechanisms, and ethical concerns must be considered.Through personalized medicine, combination therapies, and global collaboration, there is still the possibility of improving treatment outcomes in the future.As well as optimizing T. gondii-based therapies using nanotechnology, breakthroughs may be possible in this field.Cancer patients worldwide can find hope and healing by using this field's determination, collaboration, and innovation to find a cure for tumor-associated immunosuppression despite hurdles.AUTHOR CONTRIBUTIONS Narges Lotfalizadeh: Data curation (equal); formal analysis (equal); investigation (equal); writingoriginal draft (equal); writingreview and editing (equal).Soheil Sadr: Conceptualization (equal); data curation (equal); formal analysis (equal); investigation (equal); methodology (equal); validation (equal); visualization (equal); writingoriginal draft (equal); writingreview and editing (equal).Solmaz Morovati: Data curation (equal); investigation (equal); writingoriginal draft (equal); writingreview and editing (equal).Mohammadhassan Lotfalizadeh: Data curation (equal); methodology (equal); writingoriginal draft (equal); writingreview and editing (equal).Ashkan Hajjafari: Data curation (equal); software (equal); visualization (equal); writingoriginal draft (equal).Hassan Borji: Conceptualization (equal); methodology (equal); project administration (equal); resources (equal); supervision (equal); visualization (equal); writingreview and editing (equal).